Association of IL-10 and IL-10Rβ gene polymorphisms with graft-versus-host disease after haematopoietic stem cell transplantation from an HLA-identical sibling donor

Extensive allelic matching in the human leukocyte antigen (HLA) genes between recipient and donor is regarded as a prerequisite for good clinical success of allogeneic haematopoietic stem cell transplantation (HSCT) [1]. Graft-versus-host disease (GvHD) can not be totally avoided even with fully HLA-matched sibling donors or by using current GvHD prophylaxis, indicating complex and multifactorial nature of GvHD. This has lead into search of novel genetic factors, in addition to the HLA genes, to prevent and predict the occurrence of GvHD. Genes for killer cell immunoglobulin like receptors (KIR), KIR ligands and minor histocompatibility antigens as well as several cytokines and cytokine receptors have obtained a considerable interest [2–4].

Cytokine interleukin 10 (IL-10) is a good functional candidate for GvHD-related gene [5, 6]. Previous studies have shown that patient or donor IL-10 genotypes separately associate with both acute and chronic GvHD [6–9]. IL-10 is produced by a variety of different cells, of both haematopoietic and non-haematopoietic lineages [10]. IL-10 is usually regarded as a potent suppressor of the immune responses and hence it is thought to be useful in preventing GvHD. However, it has also been shown to have some immunostimulatory effects [11–13]. Interleukin 10 receptor beta (IL-10Rβ) is also expressed on several cell types, and its role varies depending on the cell type it is expressed on [14]. In haematopoietic cell lineages IL-10Rβ functions as a part of IL-10 receptor together with IL-10 receptor alpha (IL-10Rα). IL-10Rα, the IL-10 specific part of the receptor complex, is only expressed on haematopoietic cells [5]. Thus in haematopoietic cells IL-10Rβ is mediating the IL-10 signal. In many non-haematopoietic cell lineages IL-10Rβ functions as a part of a receptor for other cytokines [14].

To address the role of IL-10 and IL-10Rβ in HSCT we here report the effects of polymorphisms in these genes on the incidence of acute and chronic GvHD in 309 Finnish HSCT recipients who had an HLA identical sibling donor. In particular, we addressed the questions of interplay of patient- and donor-specific factors of IL-10 and IL-10Rβ genes.

In the present study we analysed the role of IL-10 and IL-10Rβ SNPs on the incidence of acute and chronic GvHD after HSCT between HLA-identical siblings. Previously published studies on interaction of IL-10 and HSCT have found association between IL-10 polymorphism and GvHD outcome [6, 8, 9, 15–18]. In these studies the results predominantly rely on polymorphisms of either recipient or donor. Here we addressed the question also by studying the association of the SNPs with GvHD in biologically more meaningful context; that is analysing simultaneously both donor and patient genotypes.

In our analysis patient IL-10 rs1800872 genotype A/A was associated with worse acute GvHD outcome (Additional file 1 and Table 3). Similar result was also observed with rs1800896 and rs1800872 genotype combination AA/AA. Also patient IL-10Rβ rs28341676 genotype affected the risk of developing GvHD. Patient IL-10Rβ rs28341676 A/A predisposed to and A/G protected from grades III–IV acute GvHD. Donor IL-10 rs1800896 and rs1800872 or IL-10Rβ rs28341676 polymorphisms did not have any clear effect on the GvHD outcome (Additional file 2).

Because the cells of the graft are mainly haematopoietic cells, the effect of IL-10Rβ polymorphism on the donor side is restricted to recognition of IL-10. Since most of the patient's original haematopoietic cells are eliminated, the effect of IL-10Rβ on patient side is largely restricted to the signalling of other cytokines where the IL-10Rβ also functions as a part of the receptor complex. Cytokines known to date to utilise IL-10Rβ are IL-10, IL-22, IL-26, IL-28A, IL-28B and IL-29 [14]. Since donor IL-10Rβ had no significance on GvHD predisposition, the effects of IL-10Rβ polymorphism would seem to rely largely on functions of these other cytokines utilizing IL-10Rβ in their signalling.

When analysing both the patient and donor IL-10 genotypes the results showed a trend for lower risk of developing GvHD when both had similar IL-10 production levels estimated from genotypes (Additional file 3). Similar results were also seen by Bertinetto et al [18]. In their study the presence of IL-10 rs180087 G allele in both patient and donor was associated with a trend for lower risk of developing GvHD. The rationale for this is the fact that IL-10 is a strong regulator of immune system and it might be that either higher or lower production level of the protein as compared to original one would not be optimal for the patient's immune system. The effect of patient IL-10Rβ was also amplified when analyzed together with IL-10 production level. IL-10Rβ A/A strongly predisposed to acute GvHD when patient and donor IL-10 production level differed, but protected from chronic GvHD when patient and donor had similar IL-10 production level (Additional file 3 and Table 3).

We were not able to reproduce most of the significant results of Lin et al [7] with our study material. In our results the donor IL-10Rβ had no significant effect on the GvHD even in combination with IL-10 polymorphisms. Also, patient IL-10 rs1800872 A/A predisposed to acute GvHD (Additional file 1 p = 0.031, OR = 3.83). However, as Lin et al also observed, no acute GvHD cases were present among those three pairs with the combination of patient IL-10 rs1800872 A/A and donor IL-10Rβ rs28341676 G/G (Table 2). Compared to our results, in Lin et al study the frequency of IL-10 rs1800872 A allele was higher. This might in part explain the striking difference between our results. The rs1800872 A allele is often associated with lower risk of developing GvHD, but there are also studies which show similar association as we found [6, 18]. It is possible that the rs1800872 is not it self affecting to the risk of GvHD, but is in linkage disequilibrium with the actual effector. In different populations this linkage might vary and cause the discrepancy in the results.

In analyses of IL-10Rβ rs28341676 the results differed between acute and chronic GvHD. Patient IL-10Rβ A/A predisposed to acute GvHD and seemed to protect from chronic GvHD. The different effect of IL-10Rβ polymorphism between acute GvHD and chronic GvHD and overall weaker results in chronic GvHD can be understood by the immunologic differences between acute and chronic GvHD. During the period of which acute GvHD is observed, the immune system is just starting to reconstitute anew after the HSCT. Also, many of the effector cells had been developed within the donor. In many cell lineages their numbers start rising only well after the usual 100 days time limit of acute GvHD diagnosis. More cells of donor origin are developing within the recipient during chronic GvHD. The immune system is more mature and complex than in the newly transplanted patient [19]. Notably, amounts of regulatory cells rise only relatively late in immune reconstitution [20]. The full reconstitution of immune system after transplantation may take years. Hence it is not surprising that IL-10 and IL-10Rβ polymorphisms have less effect in chronic GvHD than in acute GvHD. This could also explain the opposing effect of IL-10Rβ polymorphism in acute GvHD and chronic GvHD.

We are fully aware of the possible problems in analyzing all the transplantations as a single group. Grafting peripheral blood stem cells instead of bone marrow cells predisposes to higher incidence of chronic GvHD. Also pre-treatment conditioning, fully myeloablative versus reduced intensity conditioning, highly affects the complications after HSCT. To address this issue we performed logistic regression multivariate analysis to test the results of univariate analyses in the context of known GvHD affecting factors; disease status, gender match, graft origin, pre-transplantation conditioning and aGvHD as a risk factor for cGvHD. All the statistically significant results from univariate analyses remained significant in the multivariate analyses (Table 3). The p-values in this study were not corrected for multiple comparisons. These results need to be validated in other cohorts and further studies are also needed to clarify the relationships between IL-10 and IL-10Rβ.

We didobserve synergistic effect between IL-10 and IL-10Rβ in predisposition to GvHD. The effect of IL-10Rβ polymorphism was strengthened when it was analysed together with IL-10 polymorphisms. In our samples similarity of IL-10 production level was associated with lower incidence of both acute and chronic GvHD. Different effect of IL-10Rβ genotype in acute GvHD and chronic GvHD could be explained by the developing immune system, arise of new regulatory cells and passing cytokine storm associated with the transplantation.

JS, HT and JP: Research and Development, Finnish Red Cross Blood Service, Helsinki, Finland

LV: Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland

Address for correspondence: Dr Jukka Partanen, Research and Development, Finnish Red Cross Blood Service, Kivihaantie 7, 00310 Helsinki, Finland. jukka.partanen@bts.redcross.fi